Dynamin, actin and microtubules: cytoskeletal crosstalk in podocytes

项目总结/摘要 慢性肾脏疾病的全球流行正在以惊人的速度发展。仅在美国， 肾小球性肾病影响着大约2000万人，在过去的两年里，这个数字大约翻了一番。 几十年与肾脏有关的疾病正在迅速避开目前可用的治疗选择和资源。 足细胞是肾小球内独特的细胞，其具有由多个细胞组成的复杂组织。 细胞体，微管驱动的膜延伸（初级过程），肌动蛋白细胞骨架驱动的 膜延伸（足突）。由于足细胞的细胞骨架由三种不同的 肌动蛋白丝，微管和中间丝-它们的细胞骨架必须协调 调节足细胞以实现其复杂的细胞功能。细胞骨架元素之间的协调是 通过信号传导途径实现，这涉及常见的调节因子，如Rho GTP酶以及 巨大的GT3发动机。我们已经表明，发动蛋白直接调节足细胞中的肌动蛋白细胞骨架。 我们的新发现表明，发动蛋白也调节微管-特别是发动蛋白形成螺旋 围绕着微管。发动蛋白螺旋表现出非常高的GTP水解速率，这反过来抑制了 通过降低GTP的局部浓度来抑制微管聚合。需要高浓度的GTP 因为微管蛋白使用GTP结合来引发其聚合成 微管此外，发动蛋白螺旋对GTP的高水解率也使微管收缩成一个小的螺旋。 小半径，从而使微管不稳定并引发微管灾难。因此这两 GTP酶，发动蛋白和微管蛋白，受其附近GTP的可用性和发动蛋白的能力调节， 在微管周围寡聚成螺旋。发动蛋白直接影响微管动力学的能力 肌动蛋白的表达表明，动力蛋白可能是这两种细胞骨架蛋白在足细胞中的一种 用来互相交流。在《特定目标1》中，我们使用真实的时间单分子成像来破译 动力蛋白-微管和动力蛋白-肌动蛋白相互作用的机制细节。在具体目标2中，我们使用一个面板， 不同的发动蛋白突变体，足细胞培养，使用纯化蛋白的生物化学和电子显微镜 研究发动蛋白在调节足细胞微管中的作用，以及微管如何影响足细胞的功能。 局部粘连和应力纤维的形成。在具体目标3中，我们通过使用动物来测试我们的假设。 我们的新型动力蛋白特异性小鼠模型和HIV相关肾病（HIVAN）小鼠模型。我们 测试具有寡聚化倾向并预期促进微管形成的发动蛋白突变体是否 解聚可以通过使用微管药物来抵消足细胞损伤， 微管聚合鉴于微管在胞质分裂中的重要作用，我们还将研究微管在胞质分裂中的作用。 微管药物对HIVAN中观察到的足细胞增殖的影响。

Project Summary/Abstract The global epidemic of chronic kidney disease is progressing at an alarming rate. In the United States alone, glomerular kidney diseases affect some 20 million people, and this number has roughly doubled in the last two decades. Kidney-related diseases are rapidly eluding current available treatment options and resources. Podocytes are unique cells within the kidney glomerulus, which have a complex organization consisting of a cell body, microtubule-driven membrane extensions (primary processes), and actin-cytoskeleton driven membrane extensions (foot processes). Since the podocyte cytoskeleton is composed of three distinct elements - actin filaments, microtubules, and intermediate filaments - their cytoskeleton must be coordinately regulated for podocytes to fulfill their complex cellular function. Coordination between cytoskeletal elements is achieved through signaling pathways, which involve common regulators such as Rho GTPases as well as the large GTPase dynamin. We have shown that dynamin directly regulates the actin cytoskeleton in podocytes. Our novel findings suggest that dynamin also regulates microtubules – specifically that dynamin forms helices around microtubules. Dynamin helices exhibit very high rates of GTP hydrolysis, which in turn inhibit microtubule polymerization by lowering the local concentration of GTP. A high GTP concentration is required for efficient tubulin oligomerization since tubulin uses GTP binding to initiate its polymerization into microtubules. Additionally, high rate of GTP hydrolysis by dynamin helices also constricts microtubules to a small radii, thus destabilizing microtubules and initiating microtubule catastrophe. Therefore, these two GTPases, dynamin and tubulin, are regulated by the availability of GTP in their vicinity and dynamin's ability to oligomerize into helices around microtubules. The ability of dynamin to directly affect dynamics of microtubules and actin suggests that dynamin might be one of the proteins in podocytes that these two cytoskeletal proteins use to communicate with each other. In Specific Aim 1 we use real time single-molecule imaging to decipher mechanistic details of dynamin-microtubule and dynamin-actin interactions. In Specific Aim 2 we use a panel of diverse dynamin mutants, podocyte cell culture, biochemistry using purified proteins, and electron microscopy to investigate the role that dynamin plays in regulating microtubules in podocytes, and how microtubules affect the formation of focal adhesions and stress fibers. In Specific Aim 3 we test our hypothesis in animals by using our novel dynamin-specific mouse model, and a mouse model of HIV-associated nephropathy (HIVAN). We test whether a dynamin mutant that has the propensity to oligomerize and is expected to promote microtubule depolymerization can counteract podocyte injury by using microtubule drugs that either promote or inhibit microtubule polymerization. Given the essential role of microtubules in cytokinesis, we will also examine the effect of microtubule drugs on podocyte proliferation observed in HIVAN.